Heterogeneous expression of extracellular matrix molecules in the red nucleus of the rat.

Previous studies in our laboratory showed that the organization and heterogeneous molecular composition of extracellular matrix is associated with the variable cytoarchitecture, connections and specific functions of the vestibular nuclei and two related areas of the vestibular neural circuits, the inferior olive and prepositus hypoglossi nucleus. The aim of the present study is to reveal the organization and distribution of various molecular components of extracellular matrix in the red nucleus, a midbrain premotor center. Morphologically and functionally the red nucleus is comprised of the magno- and parvocellular parts, with overlapping neuronal population. By using histochemical and immunohistochemical methods, the extracellular matrix appeared as perineuronal net, axonal coat, perisynaptic matrix or diffuse network in the neuropil. In both parts of the red nucleus we have observed positive hyaluronan, tenascin-R, link protein, and lectican (aggrecan, brevican, versican, neurocan) reactions. Perineuronal nets were detected with each of the reactions and the aggrecan showed the most intense staining in the pericellular area. The two parts were clearly distinguished on the basis of neurocan and HAPLN1 expression as they have lower intensity in the perineuronal nets of large cells and in the neuropil of the magnocellular part. Additionally, in contrast to this pattern, the aggrecan was heavily labeled in the magnocellular region sharply delineating from the faintly stained parvocellular area. The most characteristic finding was that the appearance of perineuronal nets was related with the neuronal size independently from its position within the two subdivisions of red nucleus. In line with these statements none of the extracellular matrix molecules were restricted exclusively to the magno- or parvocellular division. The chemical heterogeneity of the perineuronal nets may support the recently accepted view that the red nucleus comprises more different populations of neurons than previously reported.

Extracellular matrix molecules exhibit unique expression pattern in the climbing fiber-generating precerebellar nucleus, the inferior olive.

Extracellular matrix (ECM) accumulates around different neuronal compartments of the central nervous system (CNS) or appears in diffuse reticular form throughout the neuropil. In the adult CNS, the perineuronal net (PNN) surrounds the perikarya and dendrites of various neuron types, whereas the axonal coats are aggregations of ECM around the individual synapses, and the nodal ECM is localized at the nodes of Ranvier. Previous studies in our laboratory demonstrated on rats that the heterogeneous distribution and molecular composition of ECM is associated with the variable cytoarchitecture and hodological organization of the vestibular nuclei and may also be related to their specific functions in gaze and posture control as well as in the compensatory mechanisms following vestibular lesion. Here, we investigated the ECM expression pattern in the climbing fiber-generating inferior olive (IO), which is functionally related to the vestibular nuclei. By using histochemical and immunohistochemical methods, the most characteristic finding was the lack of PNNs, presumably due to the absence of synapses on the perikarya and proximal dendrites of IO neurons. On the other hand, the darkly stained dots or ring-like structures in the neuropil might represent the periaxonal coats around the axon terminals of olivary synaptic glomeruli. We have observed positive ECM reaction for the hyaluronan, tenascin-R, hyaluronan and proteoglycan link protein 1 (HAPLN1) and various chondroitin sulfate proteoglycans. The staining intensity and distribution of ECM molecules revealed a number of differences between the functionally different subnuclei of IO. We hypothesized that the different molecular composition and intensity differences of ECM reaction is associated with different control mechanisms of gaze and posture control executed by the visuomotor-vestibular, somatosensory and integrative subnuclei of the IO.

Distribution of extracellular matrix macromolecules in the vestibular nuclei and cerebellum of the frog, Rana esculenta.

The axons of transected and re-apposed vestibulocochlear nerve of the frog, in contrast to mammalian species, regenerate and establish functional contacts within their original termination areas of the vestibular nuclear complex and the cerebellum. The lack of regenerative capability of the mammalian central nervous system (CNS) is partially attributed to various extracellular matrix (ECM) molecules, such as chondroitin sulfate proteoglycans (CSPG) and tenascin-R (TN-R), which exert inhibition on axon regeneration. In contrast to these molecules, hyaluronan (HA) was reported to be permissive for CNS regeneration. Using histochemical and immunohistochemical methods, we investigated the distribution pattern of these molecules in the medial (MVN), lateral (LVN), superior and descending vestibular nuclei and the cerebellum of the frog and detected regional differences in the organization of the ECM. In the vestibular nuclear complex, pericellular condensation of the ECM, the perineuronal nets (PNNs) were recognizable in the LVN and MVN and were positive only for HA. The neuropil of the vestibular nuclei showed either a diffuse appearance with varying intensity of reactions, or dots and ring-like structures, which may represent the perinodal ECM of the vestibular fibers. In the cerebellum, indistinct PNNs that were only labeled for HA were present in the granular layer. Our findings suggest that the HA-rich, but CSPG and TN-R-free PNNs may be associated with the high degree of plasticity and regenerative potential of the amphibian vestibular system.

Osteoarthritis-like damage of cartilage in the temporomandibular joints in mice with autoimmune inflammatory arthritis.

OBJECTIVE: To study temporomandibular joint (TMJ) involvement in an autoimmune murine model of rheumatoid arthritis (RA), a disease characterized by inflammatory destruction of the synovial joints. Although TMJ dysfunction is frequently found in RA, TMJ involvement in RA remains unclear, and TMJ pathology has not been studied in systemic autoimmune animal models of RA. METHODS: Proteoglycan (PG) aggrecan-induced arthritis (PGIA) was generated in genetically susceptible BALB/c mice. TMJs and joint tissues/cartilage were harvested for histological and immunohistochemical analyses and RNA isolation for quantitative polymerase chain-reaction. Serum cytokine levels were measured in mice with acute or chronic arthritis, and in non-arthritic control animals. RESULTS: Despite the development of destructive synovitis in the limbs, little or no synovial inflammation was found in the TMJs of mice with PGIA. However, the TMJs of arthritic mice showed evidence of aggrecanase- and matrix metalloproteinase-mediated loss of glycosaminoglycan-containing aggrecan, and in the most severe cases, structural damage of cartilage. Serum levels of pro-inflammatory cytokines, including interleukin (IL)-1ß, were elevated in arthritic animals. Expression of the IL-1ß gene was also high in the inflamed limbs, but essentially normal in the TMJs. Local expression of genes encoding matrix-degrading enzymes (aggrecanases and stromelysin) was upregulated to a similar degree in both the limbs and the TMJs. CONCLUSION: We propose that constantly elevated levels of catabolic cytokines, such as IL-1ß, in the circulation (released from inflamed joints) create a pro-inflammatory milieu within the TMJ, causing local upregulation of proteolytic enzymes and subsequent loss of aggrecan from cartilage.

Selective innervation of lamina I projection neurones that possess the neurokinin 1 receptor by serotonin-containing axons in the rat spinal cord.

Axons containing serotonin descend from brainstem to spinal cord and are thought to contribute to stimulation-produced and opioid analgesia, partly by a direct inhibitory action of serotonin on projection neurones. The density of serotoninergic innervation is highest in lamina I, which contains many nociceptive projection neurones. Two sets of anatomical criteria have been used to classify lamina I projection neurones: somatodendritic morphology and presence or absence of the neurokinin 1 receptor. To test whether the strength of serotoninergic innervation of lamina I projection neurones was related to morphology or neurokinin 1 receptor expression, we used confocal microscopy to determine the density of serotoninergic contacts on 60 cells retrogradely labelled from the caudal ventrolateral medulla. The contact density on neurones with the neurokinin 1 receptor was variable, with some cells receiving heavy input and others having few contacts. However, on average they received significantly more contacts (5.64 per 1000 microm(2) plasma membrane +/- 0.47, S.E.M.) than neurones which lacked the receptor (2.49 +/- .36). Among the neurokinin 1 neurones, serotoninergic innervation density was not related to morphology. Since the majority of serotoninergic boutons in lamina I of rat spinal cord do not appear to form synapses, we carried out electron microscopy on three heavily innervated neurokinin 1 receptor-immunoreactive projection neurones. Symmetrical synapses were found at 89% of serotoninergic contacts. These results indicate that serotoninergic innervation of lamina I projection neurones in the rat spinal cord is related to expression of neurokinin 1 receptors, but not to morphology, and that (at least on heavily innervated neurones) most serotonin-containing boutons which are in contact form synapses.

Location of dye-coupled second order and of efferent vestibular neurons labeled from individual semicircular canal or otolith organs in the frog.

Vestibular nerve branches innervating the sensory epithelia of the three semicircular canals or of the three otolith organs of frogs were selectively labeled in-vitro with biocytin. Labeled afferent fibers from the semicircular canals, utricle, and lagena were encountered in each of the four vestibular nuclei and their projections overlapped considerably. Saccular afferent fibers projected to the dorsal (acoustic) nuclei and smaller projections to the vestibular nuclei were regionally restricted. Per semicircular canal or otolith organ about equal numbers (11-14) of medium sized vestibular neurons (between 7.5 and 17 microm in diameter) were dye-coupled to afferent fibers. Most of these dye-coupled vestibular neurons were located in the lateral and descending vestibular nuclei between the VIIIth and IXth nerves. The superior vestibular nucleus was relatively free of dye-coupled vestibular neurons. The location of this subpopulation of central vestibular neurons supports the notion that these neurons are part of a particular vestibulospinal pathway. In addition, from each of the canal and/or otolith organs about 3-4 efferent vestibular neurons were labeled retrogradely. These neurons (between 15 and 26 microm in diameter) were located ventral to the vestibular nuclear complex. The branching of efferent vestibular neurons was shown by the presence of neurons that were double labeled by two different fluorescent dyes applied in the same experiment to the anterior and posterior ramus of the same VIIIth nerve, respectively. The branching of these efferent neuron axons explained the presence of collaterals and terminals in the sensory epithelia of a number of untreated ipsilateral endorgans.

Organization of the motor centres for the innervation of different muscles of the tongue: a neuromorphological study in the frog.

Cobalt labelling studies on the localization and morphology of the frog's hypoglossal nucleus have revealed three subnuclei. The dorsomedial subnucleus innervates the geniohyoid, hyoglossus, genioglossus and the intrinsic tongue muscles. The ventrolateral subnucleus supplies the sternohyoid, geniohyoid, omohyoid and intrinsic tongue muscles. The intermediate subnucleus innervates the omohyoid, geniohyoid and intrinsic tongue muscles. Neurons innervating protractor, retractor and intrinsic tongue muscles differ in their soma surface area and in their dendritic arborization pattern. It is concluded that there exists a musculotopic organization in the frog's hypoglossal nucleus and that motoneurons subserving different function in tongue movements disclose characteristic morphological differences.

Projections from the nucleus isthmi to the visual and auditory centres in the frog, Rana esculenta.

The lectin Phaseolus vulgaris leucoagglutinin was injected into the nucleus isthmi (NI) in order to study its anterograde and retrograde projections in the frog. The following areas of termination could be discerned in the brainstem: (1) Each of the five subnuclei of the torus semicircularis (TOS) received fibres from the NI. The projection was the most extensive on the three main subnuclei which disclosed also retrogradely labelled neurones on the side of injections. The subependymal subnuclei contained the least number of labelled fibres. (2) Both hemispheres of the optic tectum (TO) were supplied by fibres from the NI. Labelled fibres were more numerous on the side of injections, and preterminal and terminal fibres covered columnar-like areas in layers 8 and 9. Several retrogradely labelled neurones were found in layer 6. Relatively few labelled fibres were seen on the contralateral side. They formed patch-like areas of termination in layer 9. (3) The anterodorsal (AD) and anteroventral (AV) nuclei were reciprocally inter-connected with the NI. The fibre connections were less extensive on the contralateral side. In the rhombencephalon (4) the cochlear nucleus (CN) and (5) the superior olive (SO) were also reciprocally connected with the NI on both sides, but with much weaker projection on the side contralateral to injections. (6) Only a weak anterograde labelling was observed in the contralateral NI and in the ipsilateral reticular formation.

Organization of the ambiguus nucleus in the frog (Rana esculenta).

The common root of the glossopharyngeal, vagal, and accessory nerves and the individual branches of the vagus complex were labeled with cobalt, and the organization of the ambiguus nucleus was studied. The cell column labeled through the common root extended from the upper part of the medulla to the rostral spinal cord over a distance of about 3,500 microns. The labeling of individual branches revealed four subdivisions. 1) The pharyngomotor subdivision occupied the rostral 800 microns of the cell column. It gave origin to the innervation of the pharyngeal muscles. 2) The visceromotor subdivision, consisting of small and medium-sized cells labeled by way of the visceral branches of the vagus, was found in the rostrocaudal extent of the medulla. 3) the laryngomotor subdivision extended in the obex region over a distance of more than 1,000 microns. It supplied the sphincter muscles of the larynx. The dilator laryngeal muscle was represented in the rostral part of the visceromotor subdivision. 4) The accessory nerve subdivision was located in the lower medulla and the rostral spinal cord. From the results, the following conclusions are drawn. 1) The basic organization of the frog ambiguus nucleus is comparable to that of the rat, differences in nuclear organization reflecting differences in peripheral structures. 2) The cytoarchitectonic structure of the four subdivisions innervating different peripheral targets characteristically differ from each other. 3) On the basis of its characteristic neuronal morphology, the accessory nerve nucleus is regarded as an independent structure.

Sub-population of capsaicin sensitive primary afferent neurons in thoracic, lumbar and sacral dorsal root ganglion in young rats revealed by stimulated cobalt uptake.

The number of dorsal root ganglion (DRG) neurons, the relative number of capsaicin sensitive DRG cells and the diameter distribution of these neurons were investigated in the thoracic 11, lumbar 5 and sacral 1 DRG ganglia in young rats. The capsaicin sensitivity of DRG cells was shown by the stimulated cobalt uptake technique in in vitro conditions. Cobalt labelled and non-labelled neurons were counted using the dissector method. Our results show that the total number of DRG cells in the Th11, L5 and S1 segments (4200-6500 per ganglion) were not significantly different from each other and about 8% of these cells were capsaicin sensitive in the segments studied. These findings also show that the capsaicin sensitive DRG cells belong to the small diameter DRG cell population. Control experiments on neonatally capsaicin injected animals indicate that the capsaicin stimulated cobalt uptake provides selective and specific staining of capsaicin sensitive DRG neurons.

Connections of the torus semicircularis and oliva superior in the frog, Rana esculenta: a Phaseolus vulgaris leucoagglutinin labeling study.

The afferent and efferent connections of the frog principal nucleus (TP) of torus semicircularis (TOS) and superior olive (SO) were examined by employing the anterograde and retrograde transport patterns of Phaseolus vulgaris leucoagglutinin (PHA-L). After injecting the tracer into these nuclei it was found that the TP projected to the ipsilateral posterior and central thalamic nuclei, all subdivisions of the bilateral TDS and the ipsilateral nucleus isthmi (NI). In the rhombencephalon the projection was restricted mainly to the contralateral SO and the cochlear nucleus (CN). Retrogradely labeled cells were found in most of the areas that contained anterogradely labeled terminals. The termination areas of the SO fibers were similar to the projections of fibers of TP origin in the diencephalic and in the mesencephalic auditory centers. A strong projection was followed into the contralateral SO; the CNs received fibers at both sides. Caudally to the SO the reticular formation, the spinal nucleus of the trigeminal nerve, the solitary nucleus and the dorsal column nuclei were supplied by the fibers of the SO origin. Retrogradely labeled cells were found in the TOS, tegmental nuclei, solitary nucleus, dorsal column nuclei and in the spinal nucleus of the trigeminal nerve. Our results indicate that the frog auditory pathway is more complex at the level of the secondary and tertiary fiber projections than has been previously recognized.

Investigation of the dendritic geometry of brain stem motoneurons with different functions using multivariant statistical techniques in the frog.

We give an account of an effort to make quantitative morphological distinctions between motoneurons innervating functionally different muscles in the trigeminal and facial motor nuclei of the frog. Six groups of neurons were considered in the two nuclei on the basis of their peripheral targets. One group consisted of neurons (n = 7) innervating the levator bulbi muscle, which separates the orbital cavity from the oral cavity. In the second, third and fourth groups, motoneurons (n = 27) innervating jaw closer muscles (temporalis, masseter, pterygoideus) were studied. Neurons (n = 6) innervating the submaxillary muscle comprised the fifth group. This muscle forms the muscular floor of the mouth. It is active in deglutition and contributes to the opening of the mouth. The sixth group is formed by neurons of the facial nucleus (n = 7), which innervate the depressor mandibulae muscle. This is the main opener of the mouth. Neurons were selectively stained by cobalt labelling through the muscle nerves and the morphometric values of successfully labelled neurons were fed into a IBM AT 386 computer through a digitizing tablet for three-dimensional reconstruction. Four neurons labelled directly through the motor root of the trigeminal nerve but innervating unidentified muscles were added to the investigation. Two sets of quantitative measurements were taken from the neurons. In the first set (neurometric data), 17 quantitative variables were measured in the perikaryon and the dendritic arbor. In the second set, 15 variables concerned with the orientation and shape of the dendritic tree, the relation of the perikaryon to the dendritic tree and the spatial expansion of dendrites were measured in the three dimensions of Cartesian space (product-moment data). The data were subjected to multivariant statistical analysis. First, they were partitioned with cluster analysis. The average linkage between groups algorithm and the cosine of vectors of variables, or the Pearson correlation similarity coefficients were used. Neurometric data and product-moment data were analysed separately and in combination, and six to seven clusters were considered. In each case, the majority of neurons innervating jaw closer muscles were grouped into clusters different from neurons innervating jaw opener muscles. The best separation of functionally different neurons was achieved with the neurometric data set. The groups of neurons obtained from cluster analysis were subjected to non-parametric discriminant analysis with the eight nearest-neighbour classification criterion, and the results were checked with a cross-validation technique.(ABSTRACT TRUNCATED AT 400 WORDS)

Synaptic relations of the trigeminal motoneurons in a frog (Rana esculenta).

The cobalt tracing technique was used to study synaptic relations of trigeminal motoneurons and the axon terminals of the mesencephalic trigeminal nucleus (Vmes) in a frog (Rana esculenta). Most of the small calibre Vmes terminals containing spherical synaptic vesicles occupied presynaptic position in axodendritic contacts. Axoaxonic contacts of Vmes boutons were exclusively found at the postsynaptic site. The synaptic coverage of the trigeminal motoneuron dendritic tree increased somatofugally along the dendrites. Monosynaptic contacts between the trigeminal motor and mesencephalic nuclei occur very rarely indicating that this type of activation does not play a significant role in the frog.

Motoneurons differ in size and peripheral target in the trigeminal and facial nuclear complex of the frog.

The cobalt labelling technique was applied to different branches of the frog trigeminal and facial nerves and the representation of muscle supplied by these nerves were studied. (1) The levator bulbi muscle is innervated by a small group of neurons localised in its rostromedial part of the trigeminal motor nucleus (nV). (2) The jaw closer muscles are represented in the rostral two thirds of the nV. (3) Muscles of the floor of the mouth, which contribute to the jaw opening, and the depressor mandibulae muscle are innervated from the caudal one third of the nV and from the facial motor nucleus, respectively. There were distinct differences in the shape and size of perikarya innervating these three functionally different muscle groups.

Cobalt uptake enables identification of capsaicin- and bradykinin-sensitive subpopulations of rat dorsal root ganglion cells in vitro.

A novel modification of the stimulated cobalt uptake technique has been used to identify rat dorsal root ganglion cells expressing capsaicin and bradykinin receptors. The technique involves incubating intact dorsal root ganglia in vitro in a modified Krebs solution in which cobalt chloride has been substituted for calcium. Activation of dorsal root ganglion cells by capsaicin or bradykinin in the presence of the cobalt ions results in cobalt influx into the excited cells. Histochemical methods were then used to visualize the intracellular accumulation of cobalt, and labelled cells were counted and characterized. Capsaicin (2 microM) or bradykinin (500 nM) applied for 20 min induced cobalt uptake in 13.8 +/- 0.6 and 9.6 +/- 0.5% of neuronal profiles in dorsal root ganglia (L4), respectively, a significantly larger number than stained in control ganglia (in the absence of agonists: 1.8 +/- 0.7%). The longest diameter of the soma of stained dorsal root ganglion cells following capsaicin and bradykinin perfusion were significantly different from each other and from the non-labelled population (17.5 +/- 0.7 and 24.5 +/- 0.2 microns for capsaicin; 23.2 +/- 0.9 and 25.5 +/- 0.4 microns for bradykinin; labelled and non-labelled cells, respectively). The distribution of cell diameters revealed that while capsaicin-sensitive cells were exclusively small-sized, bradykinin-sensitive cells were predominantly small and medium sized. The selective bradykinin-2 receptor antagonist HOE-140 (5.0 microM) blocked the bradykinin-induced staining (2.16 +/- 0.02%) but not that of capsaicin. The bradykinin-1 agonist [des-Arg9]-bradykinin did not induce any significant increase in stained cells over the control number (2.2 +/- 0.7%).(ABSTRACT TRUNCATED AT 250 WORDS)

The efferent system of cranial nerve nuclei: a comparative neuromorphological study.

A number of inconsistencies and controversies are inherent in the classification of cranial nerve nuclei based on the concepts of the various head-theories. The assumption of head segmentation, which is common to these theories, serves as the basis for designating the dorsomedial nuclei as the somatomotor column, although they innervate striated muscles of a viscus and a specific sense organ. The ventrolateral nuclei are called the specific visceromotor column; they innervate striated muscles in the branchiogenous area, but many of these muscles insert on skeletal elements. A series of comparative neuromorphological studies investigating the dendritic arborization pattern and axonal trajectory in the frog, lizard, and rat suggests a much more delicate classification in which nine morphologically and functionally different neuron groups can be discerned: 1. The hypoglossal nucleus appears coincidentally with the muscular tongue in amphibia. The spindle-shaped perikaryon, the bipolar dendritic arborization, and the straight ventral trajectory of the axon are characteristic morphological features in all three animal species investigated. 2. The oculomotor, trochlear, and abducens nuclei present a remarkably conservative topography and organization in all vertebrates with a moving eye. With their oval-shaped or polygonal perikarya and radiating dendritic arborization, these neurons distinctly differ from hypoglossal neurons. The ipsilateral axons follow a straight ventral course, the contralateral axons form a dorsal loop before crossing the midline, and the crossing is not consequence of neuron migration to the contralateral side. 3. The accessory abducens nucleus is present in tetrapods except apes and human. The elongated perikaryon and the dorsoventral dendritic orientation distinctly distinguish these neurons from other cranial motoneurons, the nucleus is found in the lateral part of the reticular formation. The neurons differentiate in situ, they do not migrate from the main abducens nucleus. 4. In the submammalian trigeminal and facial nuclei, two basic neuron types can be distinguished on the basis of their morphology. The first type is larger and accumulates in the rostral part of the trigeminal nucleus. This type innervates the jaw closer muscles. The second type is found in the caudal part of the trigeminal nucleus and in the facial nucleus. These neurons innervate the muscular floor of the mouth and the facial contingent supplies the jaw opener muscle. A very characteristic feature in the axonal trajectory is an initial medial course and a hairpin turn, or dorsal loop, at the lateral aspect of the medial longitudinal fasciculus. In addition to the two types of neurons, there is a third type in the frog trigeminal nucleus. This innervates an orbital muscle.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of the oculomotor and trochlear nuclei in the Xenopus toad.

The development of the oculomotor and trochlear nuclei (nIII and nIV) was studied with the horseradish peroxidase (HRP) and the cobalt labelling techniques in Xenopus laevis tadpoles. The earliest labelling of the oculomotor neuroblasts was observed at stage 32. The ipsi- and contralateral nuclei were found in two distinct groups on either side of the brainstem and the oculomotor commissure formed by crossing axons was present at this early stage. The fusion of the two nuclei began at the late larval stage when the axonal outgrowth had been presumably completed. The trochlear neuroblasts could be first labelled at stage 39 when the position of the nucleus and axonal pathway was similar to the adult form.

Development of the abducens nuclei in the Xenopus laevis.

The development of the main (nVI) and the accessory abducens (nVIa) nuclei was studied with the horseradish peroxidase and cobaltic-lysine labeling techniques in Xenopus laevis tadpoles. In earliest labeling was obtained at stage 39, and neuroblasts of both nuclei formed two separate groups according to their definitive positions in relation to other rhombencephalic structures in this young age of development. Conspicuous morphological differences were observed between the two nuclei: the accessory abducens neuroblasts were twice as big as the abducens neuroblasts and the characteristic nVIa 'knee' was present from this time of the first successful labeling. The two different dendritic arborization patterns, which clearly distinguished the abducens neurons from the accessory abducens neurons, gradually developed in tadpoles. It is suggested that the form and position of abducens and accessory abducens neurons are determined at a prefunctional stage, probably before the beginning of axonal outgrowth, and neurobiotaxis may not play the role attributed previously in the differentiation of these two nuclei.